DESCRIPTION: (Applicant's Abstract) The long term goal of this research is to elucidate mechanisms regulating an intracellular Ca channel (the InsP3-gated Ca channel). The InsP3-gated Ca channel is found in a wide variety of cells, and plays a key role in the rise of intracellular Ca concentration following hormonal/neurotransmitter stimulation. The concentration of InsP3 needed to stimulate Ca release in neurons such as the cerebellar Purkjnje cell, however, is at least 50 times higher than that needed for peripheral cells. Recently, we have obtained evidence for the presence of an endogenous inhibitor of the InsP3 receptor, which may explain this low InsP3 sensitivity of neurons. We call this inhibitor "IRI" (for InsP3 Receptor Inhibitor), and have obtained preliminary data indicating that it decreases the sensitivity of the channel complex to InsP3, both in vitro and in living cells. Our preliminary data suggest that this inhibitor IRI is a sulfated, low molecular oligosaccharide (1300 Da), though further studies (aimed at elucidating its sequence) are underway. We have also obtained data indicating that IRI differs substantially from heparin, which is secreted from cells. We hypothesize that high levels of IRI in some neuronal cells may contribute to their low InsP3 sensitivity compared to peripheral cells, and have obtained preliminary data in support of this hypothesis. Further studies, however, are needed to critically/quantitatively test the latter hypothesis. Efforts will also be directed at determining the rates of synthesis and degradation of IRI. We hypothesize that the synthesis of IRI increases secondary to either a morphological change in neurons (i.e., during the formation of dendrites), and we will test this hypothesis directly using neuroblastoma before and after differentiation (i.e., before and after formation of neuronal processes). Efforts will also be directed at the localization of IRI, which we presume is in the cytoplasm. The possibility that IRI is secreted will also be examined. Lastly, the specificity of IRI will be assessed. Most cells express more than one isoform of the InsP3 receptor, raising the possibilty that the InsP3 sensitivity of a cell may dependent not only on the intracellular concentration of IRI, but also the relative abundance of the various InsP3 receptor isoforms. Our preliminary data suggest that the type 3 InsP3 receptor isoform has a much lower sensitivity to IRI than either the type 1 or type 2 InsP3 receptor isoform, though further studies are needed. The proposed study will therefore provide valuable insights into the structure, function, regulation, and specificity of IRI as an endogenous inhibitor of neuronal InsP3 gated Ca channels, and thus may provide a foundation for studies aimed at abnormalities underlying altered intracellular Ca regulation in pathological states